1. Determination of Alkaline Phosphatase activity
Effect of substrate concentration and enzyme inhibitor on enzyme activity
Determination of Alkaline Phosphatase activity

2. Introduction:
Phosphatases are enzymes which catalyze the splitting off phosphoric acid from nonphosphoric esters.
Two common types are estimated in serum:
Alkaline Phosphatase (optimum pH 10)
Acid Phosphatase (optimum pH 5 – 6)

3. Alkaline Phosphatase
Purified forms from different sources undergo 3 types of activity:
1- Hydrolytic
R-O- P + HOH ROH + H3PO4
2- Phosphotransferase
R-O- P + R`-OH R- OH + R`- O- P
3- Pyrophosphatase:
R-O- P -O- P -O- R` + HOH R-O- P + R`-O- P

4. Sources: Osteoblasts in the bone Bile canaliculi in liver
Small intestinal epithelium
Proximal tubules in the kidney.
The placenta
Lactating breasts
In all these sites, it seems to be involved in the transport of phosphate across membrane
ALP of normal serum is mainly derived from liver (the bone isozyme is absent)

5. ALP requires metal ions : Inhibitors of ALP:
Mg2+, Zn2+ and to a lesser extent Mn2+
Inhibitors of ALP:
Cu2+, Hg2+, EDTA (chelating Mg2+), Phosphate & some amino acids e.g. L-phenylalanine

6. Principle of the test:
ALP from human serum will hydrolyze the artificial substrate, disodium phenyl phosphate to phenol which will react with 4-aminoantipyrine in the alkaline, oxidizing agents giving a red purple color which can be measured at 520 nm

7. Objective of the test
In this experiment we will investigate the effect of changing substrate concentration on enzyme activity in presence and in absence of the inhibitor (inorganic phosphate) to identify the type of inhibition.

8. Into 10 test tubes, add the following (in ml):
PROCEDURE:
Into 10 test tubes, add the following (in ml):
Blank
Standard
Test Test
Without Inhibitor With Inhibitor 1 2 3 4 5 6 7 8
Buffer
1.0
Substrate -
0.25
0.50
0.75
Substrate + Inhibitor
Dist. Water
1.1
0.1
Serum sample
Mix and incubate the tubes at 37C for exactly 15 min.
NaOH N
0.8
Na HCO N
1.2
4-amino-antipyrine
Potassium ferricyanide
Mix and read the tubes against blank at 520 nm

9. Calculate ALP activity (v) for each substrate
concentration using the following formula:
ALP activity (v) = X 10
= mg of phenol produced/100 ml
serum in 15 minutes
= King Armistrong (KA) units/100 ml
O.D test – O.D control
O.D Standard – O.D blank

10. Calculation of substrate concentration:
Substrate concentration = 0.01 M
Substrate M. Wt = 254
Substrate concentration = 0.01 X 254 = 2.54
[S] = substrate conc. X volume
= 2.54 X volume

11. 1. Calculation: O.D. B = 0.0 O.D. C = 0.04
O.D. S = [S] = 2.54 X volume
Test tube
Sub.volume
(ml)
[S]
1/[S]
O.D.T
T- C
V = X 10
S – B
1/V 1
0.25
0.635
1.575 2
0.50
1.27
0.787 3
0.75
1.905
0.525 4
1.00
2.54
0.394 5 6 7 8 -I +I

12. 2. Type of inhibition: Using 1/V and 1/[S], draw the Linweaver-Burk plot to calculate the Km and Vmax of the reaction in presence and absence of the inhibitor
1/Vmax(-I) =  Vmax (-I) =
- 1/Km (-I) =  Km (-I) =
1/Vmax(+I) = Vmax (+I) =
- 1/Km (+I) = Km (+I) =

13. Non - Competitive Inhibitor
1/vi
Competitive
Inhibitor No
- 1/Km
Vmax is the same in presence of a competitive inhibitor
Competitive Inhibitor
Apparent Km is increased in presence of a competitive Inhibitor
Non - Competitive Inhibitor
1/vi
1/Vmax
- 1/Km /[S]
No Inhibitor
+ Inhibitor
1/Vmax’
1/vi
1/Vmax
- 1/Km /[S]
No Inhibitor
+ Inhibitor
Uncompetitive Inhibitor

14. 3. Calculation of the percentage inhibition:
V Vi
% inhibition = X 100
V where V = rate without inhibitor, Vi = rate with inhibitor
[S] V Vi
% inhibition
0.635
1.27
1.905
2.54
4. Does the % inhibition change as [S] increase?
Do the results of this calculation confirm your
conclusions as to the type of inhibition?